Astaxanthin protects ARPE-19 cells from oxidative stress via upregulation of Nrf2-regulated phase II enzymes through activation of PI3K/Akt.

Purpose Oxidative stress on retinal pigment epithelial (RPE) cells is thought to play a crucial role in the development and progression of age-related macular degeneration. Astaxanthin (AST) is a carotenoid that shows significant antioxidant properties. This study was designed to investigate the protective effect of AST on ARPE-19 cells against oxidative stress and the possible underlying mechanism. Methods ARPE-19 cells exposed to different doses of H2O2 were incubated with various concentrations of AST and cell viability subsequently detected with the (4-[3-[4-iodophenyl]-2–4(4-nitrophenyl)-2H-5- tetrazolio-1,3-benzene disulfonate]; WST-1) assay. The apoptosis rate and intracellular levels of reactive oxygen species (ROS) were measured with flow cytometry. NAD(P)H quinine oxidoreductase 1 (NQO1), hemeoxygenase-1 (HO-1), glutamate-cysteine ligase modiﬁer subunit (GCLM), and glutamate-cysteine ligase catalytic subunit (GCLC) expression were examined with real-time PCR and western blotting. The nuclear localization of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) protein and the expression levels of cleaved caspase-3 and protein kinase B proteins were evaluated with western blotting. Results AST clearly reduced H2O2-induced cell viability loss, cell apoptosis, and intracellular generation of ROS. Furthermore, treatment with AST activated the Nrf2-ARE pathway by inducing Nrf2 nuclear localization. Consequently, Phase II enzymes NQO1, HO-1, GCLM, and GCLC mRNA and proteins were increased. AST inhibited expression of H2O2-induced cleaved caspase-3 protein. Activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathway was involved in the protective effect of AST on the ARPE-19 cells. Conclusions AST protected ARPE-19 cells against H2O2-induced oxidative stress via Nrf2-mediated upregulation of the expression of Phase II enzymes involving the PI3K/Akt pathway.

Cell culture: ARPE-19 cells (from ATCC cell line) were cultured in Dulbecco's modified Eagle medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 100 μg/ml of streptomycin, and 100 U/ml of penicillin. The cells were cultured at 37 °C in a humidified 5% CO 2 atmosphere, and the medium was changed every other day. The cells were grown to an appropriate density and used at passage 10-15. Measurement of cell viability: Cell viability was determined with the sulfonated tetrazolium salt WST-1. The measurement depends on the ability of viable cells to cleave tetrazolium salts by mitochondrial dehydrogenases. Briefly, cells were plated in 96-well microplates at a density of 5×10 4 cells/well. After incubation for 24 h at 37 °C, cells were treated with 0, 5, 10, or 20 µM AST for 6 h, 12 h, and 24 h at 37 °C. The cells were then treated with 200 or 400 μM H 2 O 2 for 24 h at 37 °C, WST-1 solution was added (10 µl/well), and the cells were further incubated for 3 h at 37 °C in a 5% (v/v) CO 2 atmosphere. Absorbance at 450 nm was measured with a microplate reader with a background control as the blank.
Flow cytometry analysis of cell apoptosis: The Annexin V FITC-Propidium Iodide (PI) kit was used to detect cell apoptosis. The cells were grown on a six-well plate at 1×10 5  Measurement of accumulation of intracellular reactive oxygen species: The intracellular levels of reactive oxygen species (ROS) were measured using the DCFH-DA molecular probes. Cells were incubated with 10 μM DCFH-DA for 30 min at 37 °C, then washed, and resuspended in PBS at 1×10 6 cells/ml. The cells were analyzed using flow cytometry at excitation and emission wavelengths of 488 and 525 nm, respectively. Untreated cells served as the control. The results were expressed as fluorescence intensity of dichlorofluorescein (DCF) compared with control.
Real-time polymerase chain reaction: Total RNA was extracted from the cells using TRIzol reagent (Invitrogen, Carlsbad, CA) as described by the manufacturer. Samples containing 1 μg of total RNA were reverse transcribed into cDNA with a first-strand cDNA synthesis kit (Bio-Rad, Hercules, CA) according to the manufacturer's instructions. Real-time PCR was performed with the SYBR Green PCR Master Mix kit (Applied Biosystems, Foster City, CA) on a Bio-Rad iCycler system (Bio-Rad). The fold change in the levels of NQO1, GCLM, HO-1, and GCLC between the treated and untreated cells, normalized by the level of β-actin, was determined using the following equation: fold change=2 −∆(∆Ct) , where ∆Ct= Ct (target) − Ct (β-actin) and ∆(∆Ct)=∆Ct (treated) − ∆Ct (untreated) . The primers used in this study were purchased from Invitrogen (Shanghai, China; Table 1).
Western blot analysis: After treatments, the cells were twice washed gently in ice-cold PBS and then lysed using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Haimen, China) according to the protocol described by the manufacturer. Lysates were centrifuged at 15,000 ×g for 10 min at 4 °C. Protein concentrations were determined with the Bicinchoninic Acid Protein Assay kit (Pierce, IL). Protein samples were fractionated with SDS-PAGE and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). After blocking with 5% (v/v) skim milk for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4 °C and then incubated with the corresponding horseradish peroxidase-linked secondary antibodies for 1 h at room temperature. The signals were developed using the enhanced chemiluminescence (ECL) western blotting detection reagent (Amersham Biosciences, Piscataway, NJ) and exposed to X-ray film. Densitometric analysis was performed with Quantity One software (Bio-Rad Laboratories).
Statistical analysis: Data were expressed as mean ± standard deviation (SD). All data were analyzed with one-way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test for multiple comparisons. Statistical significance was set at p≤0.05.

RESULTS
Astaxanthin prevents hydrogen peroxide-induced decrease in ARPE-19 cell viability: Oxidative damage to cells is commonly modeled using treatment with H 2 O 2 [17][18][19]. Cell viability was evaluated using WST-1 assays. We first incubated ARPE-19 cells for 24 h with different concentrations of AST and then exposed the cells to 200 µM H 2 O 2 for 24 h. Figure 2A shows a significant dose-dependent increase in cell viability; 20 µM was determined as the optimal dose for treatment with AST. To investigate whether the protective effect of AST is related to incubation time, we incubated ARPE-19 cells with AST for different lengths of time before the cells were exposed to H 2 O 2 . Figure 2B shows the cell viability increase was time-dependent and 24 h was the optimal time for AST treatment. Thus, we selected treatment with 20 µM AST for 24 h before exposure to different concentrations of H 2 O 2 . Figure 2C shows the protective effect of AST still existed when the concentration of H 2 O 2 was 400 µM; 200 µM H 2 O 2 caused an approximate 50% loss of cell viability without treatment with AST.

Protective effect of astaxanthin against hydrogen peroxideinduced cell apoptosis: H 2 O 2 induces cellular apoptosis.
To investigate whether AST protects against H 2 O 2 -induced apoptosis, ARPE-19 cells were incubated with 10 μM and 20 μΜ AST for 24 h and then exposed to 200 µM H 2 O 2 for 24 h. Cells were then stained with Annexin V⁄ PI, and the apoptosis rate was determined with flow cytometry. As shown in Figure 3, the lower right field (PI-negative, Annexin V-positive staining) indicates the apoptotic cells. Figure 3 shows a significant increase in the apoptosis rate when the ARPE-19 cells were exposed to 200 µM H 2 O 2 . Moreover, when the ARPE-19 cells were treated with different concentrations of AST, the cell attenuation of apoptosis was dose-dependent.

Astaxanthin inhibits hydrogen peroxide-induced intracellular generation of reactive oxygen species:
The ARPE-19 cells were incubated with 10 μM and 20 μΜ AST for 24 h and then treated with 200 µM H 2 O 2 for 24 h. DCF fluorescence was recorded as a measure of intracellular ROS. Figure 4 shows the levels of intracellular ROS were increased significantly in the H 2 O 2 -treated cells. However, treatment with AST resulted in a dose-dependent inhibition of intracellular production of ROS.
Effects of astaxanthin on the expression of NQO1, HO-1, GCLC, and GCLM mRNA and protein: To clarify the antioxidative mechanisms of AST against H 2 O 2 -induced cell injury in ARPE-19 cells, we examined the expression levels of Phase II enzymes, such as NQO1, HO-1, GCLM, and GCLC. Figure  5 shows treatment of ARPE-19 cells with different concentration of AST (5, 10, or 20 µM) for 24 h induced an increase in the expression of NQO1, HO-1, GCLC, and GCLM mRNAs. To expand these findings, we used western blotting to detect

Gene
Primer (5′-3′)  Involvement of the PI3K/Akt pathway in astaxanthin-induced cytoprotection against oxidative stress: The PI3K/Akt pathway has been reported to be essential in regulating the antioxidant function in the RPE cell [20]. Research indicated that Akt activation could protect RPE cells from oxidantinduced cell death [21]. It has been reported that AST could induce significant activation of PI3K in neural progenitor cells [22]. We examined Akt phosphorylation in ARPE-19 cells to determine whether the PI3K/Akt pathway is responsible for

Nuclear localization of Nrf2 protein induced by astaxanthin is regulated by the PI3K/Akt pathway:
Because Nrf2 is a crucial transcription factor for regulating the expression of endogenous antioxidant enzymes, we asked whether AST could induce Nrf2 localization in ARPE-19 cells. Figure 7 shows the results of a western blot assay indicating that treatment with 20 µM AST increased the nuclear localization of Nrf2 significantly. This increased nuclear localization of Nrf2 was blocked by LY294002 (10 µM). These results suggest AST increases the nuclear localization of Nrf2 through the PI3K/Akt pathway.

Downregulation of cleaved caspase-3 protein expression induced by astaxanthin is regulated by the PI3K/Akt pathway:
We examined the protein expression of cleaved caspase-3 to investigate the underlying mechanism of the antiapoptotic effect of AST. Figure 7 shows treatment with 200 µM H 2 O 2 markedly increased the cleavage of caspase-3. However, the increase was suppressed significantly by treatment with 20 µM AST. LY294002 (10 µM) partially reduced the ASTinduced protective effect. These results reveal the involvement of the Akt pathway in the protective effect of AST on H 2 O 2 -induced apoptosis.

DISCUSSION
This study investigated the protective effect of AST on H 2 O 2 -induced oxidative stress in ARPE-19 cells and the possible signal pathway involved. AST has been shown to have significant antihypertensive, neuroprotective, antidiabetes, and anti-obesity effects in experimental animals in vivo [23][24][25]. It has been reported that administration of AST could increase the choroidal blood flow velocity in volunteers [26]. Research indicated that supplementation with a formulation containing AST may improve visual function and help to delay progression of AMD [16]. AST is a powerful free radical scavenger and protects several types of cells from oxidative damage [8,27,28]. Although earlier studies showed that AST supplement leads to a decrease in the level of the TNF-α-induced MCP-1 protein in ARPE-19 cells, it is not known whether AST can protect ARPE-19 cells from oxidative stress [29]. Our results suggest that treatment with AST reduces H 2 O 2 -induced cell death, intracellular ROS production, and apoptosis in ARPE-19 cells, and that the mechanism by which AST induced cytoprotection could include the Nrf2-antioxidant-response element (ARE) and Akt pathways. These data indicate that AST might provide a valuable therapeutic strategy for early AMD.
Oxidative stress has been studied extensively in relation to the pathophysiology of AMD and is suggested to have a crucial role [1]. The location and physiologic function of RPE cells cause them to be constantly exposed to several ROS [3,30]. Thus, protecting RPE cells from oxidative damage is an important consideration for treating AMD. The addition of H 2 O 2 to cultured cells is a classic model used to test oxidative stress susceptibility or antioxidant efficiency in the RPE cell [18,31,32]. The present study demonstrates that treating ARPE-19 cells with H 2 O 2 results in a marked loss of viability. However, treatment with AST decreased the cell viability loss significantly. ROS-mediated cellular damage was greatly reduced with AST pretreatment in retinal ganglion cells, human neuroblastoma cells, and human lymphocytes [8,33,34]. In this study, treatment with AST resulted in much lower levels of H 2 O 2 -induced intracellular production of ROS. The results of this study, as well as many earlier studies, suggest that AST has a direct antioxidant effect by scavenging ROS from the environment [8,33,34]. Moreover, flow cytometry showed that H 2 O 2 -induced cell apoptosis in ARPE-19 cells is greatly reduced by treatment with AST. We examined the expression of cleaved caspase-3, which is known to be a stimulator of apoptosis, to further explore the effect of AST on apoptotic cell death caused by exposure to H 2 O 2 . In the present study, treatment with AST significantly reduced the expression of apoptotic protein cleaved caspase-3 induced by H 2 O 2 . These results indicate that AST protects ARPE-19 cells from H 2 O 2 -induced cell damage via its antiapoptotic and antioxidative effects.
We investigated the potential pathway involved in the protective effect of AST against oxidative stress in ARPE-19 cells. In recent years, the Nrf2-ARE pathway has been characterized as an important endogenous mechanism that attenuates oxidative stress [35,36]. Nrf2 is an obligate transcription factor that could bind to the ARE to induce the expression of Phase II enzymes [37]. In the absence of oxidant damage, Nrf2 interacts with the chaperone Keap1, whereas in an oxidant environment, Nrf2 dissociates from Keap1, activated Nrf2 translocates to the nucleus and binds to the ARE, and then Phase II enzymes are expressed [38]. NQO1 reduces quinones via a two-electron reduction, limiting the subsequent generation of ROS [39,40]. HO-1 catalyzes the ratelimiting step in heme catabolism, resulting in formation of the antioxidant bilirubin when biliverdin reductase is present [41]. GCL controls the production of glutathione, which is the most abundant endogenous antioxidant thiol [37]. The GCL holoenzyme is a heterodimer consisting of GCLC and GCLM [42]. Earlier studies indicated that activation of the Nrf2-ARE pathway and increased expression of the following Phase II enzymes could protect RPE cells against oxidative damage [37,43]. Earlier studies showed that AST and other carotenoids can activate the Nrf2-ARE pathway in cancer cells and in the rat liver [44,45]. In our study, we investigated whether AST, a well-known potent antioxidant, can increase Nrf2 nuclear localization and promote the expression of Phase II enzymes in ARPE-19 cells. As we have shown, treatment with AST can increase the nuclear localization of Nrf2 and consequently increase the expression of Phase II enzymes regulated by Nrf2, including NQO1, GCLC, GCLM, and HO-1. As shown in our results, treatment with AST effectively protects ARPE-19 cells from H 2 O 2 -induced decreases in cell viability and inhibits cell apoptosis and the intracellular production of ROS. All of these protective effects probably arise from the enhancement of the Phase II antioxidant enzyme system.
Although there is more than one pathway AST could activate, including ERK, NF-κB, and PI3K/Akt [22,29,46], we focused on Akt because it has a key role in regulating many important proteins involved in cell survival through profound antioxidant and antiapoptotic actions [4]. Earlier studies demonstrated that adding H 2 O 2 to RPE cells increased intracellular ROS production significantly and, simultaneously, caused detectable Akt activation [21]. In the present study, Akt phosphorylation was moderately enhanced in the stimulation of H 2 O 2 . Moreover, 20 μΜ AST notably increased Akt phosphorylation in ARPE-19 cells, and this effect was abolished when the cells were treated with the highly specific Akt inhibitor LY294002. These results suggest that activation of the PI3K/Akt pathway is involved in the protection of AST against H 2 O 2 -induced oxidative stress in ARPE-19 cells.
The PI3K/Akt pathway has crucial roles in modulating Nrf2-ARE-dependent protection against oxidative stress in the RPE cell [20]. Our findings indicate that the increased nuclear localization of Nrf2 induced by AST was dependent on the activation of Akt, because LY294002 decreased the AST-induced enhancement of the nuclear localization of Nrf2 via inhibition of Akt phosphorylation. Furthermore, the results of our study demonstrate that the inhibitory effect of AST on H 2 O 2 -induced cleaved caspase-3 expression was partially reversed by LY294002. Our previous study indicated that LY294002 inhibited the AST-induced cytoprotective effect on cell viability and apoptosis against H 2 O 2 (see the supplementary data). These results suggest that AST enhanced the Phase II antioxidant enzyme system and inhibited cell damage induced by H 2 O 2 through activation of the PI3K/Akt pathway (Figure 8). The results of this study indicate that AST can offer a practical approach to the oxidative damage induced by H 2 O 2 in ARPE-19 cells, which merits further investigation.
Taken together, the results of the present study provide strong evidence that treatment with AST attenuated the H 2 O 2induced oxidative stress in ARPE-19 cells and the protective mechanism was associated, at least in part, with the activation of the Nrf2-ARE and PI3K/Akt pathways. Other recent studies showed that AST protected retinal ganglion cells against H 2 O 2 -induced cell death [34]. These results are noteworthy and indicate that AST might be capable of protecting RPE cells and retinal neurons from oxidative damage. The results also suggest the possibility that administering AST is a potential therapeutic strategy for the prevention and therapy of AMD and other retinal disorders associated with oxidative stress.

APPENDIX 1. LY294002 INHIBITED THE AST-INDUCED CYTOPROTECTIVE EFFECT ON CELL VIABILITY AGAINST H2O2 IN ARPE-19 CELLS.
To access the data, click or select the words "Appendix 1." The ARPE-19 cells were treated with different concentrations of AST (0, 5, 10 and 20 µM) for 24 h and then treated with or without 10 µM LY294002 for 30 min before incubation with or without 200 µM H2O2 for 24 h. Data are shown as mean ± SD (n=6); *P <0.05 vs control. In all cases, the control is untreated RPE cells. # P <0.05 vs H2O2-induced cells without treatment with AST; §P<0.05 vs H2O2 + 20 µM AST.

APPENDIX 2. LY294002 INHIBITED THE AST-INDUCED CYTOPROTECTIVE EFFECT ON CELL APOPTOSIS AGAINST H2O2 IN ARPE-19 CELLS.
To access the data, click or select the words "Appendix 2." (A) The ARPE-19 cells were incubated with different concentrations of AST (10 µM and 20 µM) for 24 h and then treated with or without 10 µM LY294002 for 30 min before incubation with or without 200 µM H2O2 for 24 h. Flow cytometry recording shows the apoptosis rate of ARPE-19 cells. (B) Summarized data showing the rate of apoptotic cells detected by flow cytometry. Data are shown as mean ± SD (n=6); *P <0.05 vs control; # P <0.05 vs H2O2-induced cells without treatment with AST; §P<0.05 vs H2O2 + 20 µM AST.

ACKNOWLEDGMENTS
This study was supported by a grant from the National Natural Science Foundation (NSFC: 30,970,749) and partly supported by Natural Science Foundation of Heilongjiang Province (D200924). The authors confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.